Candida hemolysin-like proteins

ABSTRACT

Candida yeast such as  C. glabrata , express a hemolysin-like protein (HLP) that is structural similarity to hemolysins in other pathogens. HLP polypeptides are antigenic and are useful as vaccine components to raise a protective immune response against Candida. HLP expression is regulated by phenotype switching in  C glabrata  and coincides with the hemolytic activity of the phenotype.

BACKGROUND OF THE INVENTION

[0001]Candida albicans and C. glabrata Present a Growing Risk to PublicHealth

[0002] The yeast Candida species can exist both as a non-virulentcolonizer (commensal) and as a pathogen. Candidiasis has becomeincreasingly widespread during the last two decades, with hospitalizedand immunocompromised patients at greatest risk, and has become thesixth most common form of pathogenic infection. Systemic Candidainfections may be lethal, with a mortality rate of 50% in adults and upto 65% in infants. Colombo et al. (1999); Pacheco-Rios et al. (1997).Reviewed in Pfaller (1996). The risk of death from systemic infectionmost strongly correlates with the time between the first detectedinfection and the onset of antifungal treatment. Pacheco-Rios et al.(1997).

[0003]Candida albicans accounts for 50-70% of candidiasis cases,although a progressively larger proportion of cases are caused bynon-albicans species. C. glabrata (formerly Torulopsis glabrata) hasemerged as one of the three most common Candida species colonizinghumans (Fidel et al. (1999); Hazen et al. (1995)) and it now representsthe second most common Candida species causing blood stream infections(Pfaller et al. (1996)) and one of the most prevalent speciesresponsible for yeast vaginitis (Sobel et al. (1996); Spinillo et al.(1995)). A dramatic increase in the carriage of C. glabrata has alsobeen demonstrated in dentate individuals over 80 years of age, and theproportion of elderly individuals with dentures carrying C. glabrata inone study was found to be greater than 50%. Lockhart et al. (1999). Whatis most worrisome about the recent emergence of C. glabrata, as a majorCandida commensal and pathogen, is that it is naturally resistant toazole drug therapy. Blaschke-Hellmessen et al. (1996); Fortun et al.(1997); Hitchcock et al. (1993); Marichal et al. (1997).

[0004] Candida Pathogenicity is Enhanced by High Frequency PhenotypicChanges

[0005] The pathogenic success of Candida depends in part upon phenotypicplasticity. C. albicans, for example, exhibits a bud-hypha phenotypetransition, which occurs en masse in response to various stimuli, andprovides C. albicans with the capacity to penetrate tissue and todisseminate. Odds (1997). C. albicans also undergoes spontaneous,reversible, high frequency switching of phenotypes, which usually doesnot occur en masse. In C. albicans strain WO-1, switching occursreversibly between a phenotype characterized by white colonies and aphenotype characterized by opaque colonies. Soll (1992). In C. albicans,switching has been demonstrated to occur at higher frequencies inisolates from deep versus superficial mycoses (Jones et al. (1994)), athigher frequencies in infecting versus commensal isolates from the oralcavity (Hellstein et al. (1993)), within sites of infection (Soll et al.(1987); Soll et al. (1988)) and within sites of commensalism (Soll(1992)). Switching has also been demonstrated to regulate virulence inanimal models. Kvaal et al. (1999).

[0006] High frequency phenotypic switching in C. albicans involves thecoordinated regulation of phase-specific genes. The gene products ofseveral of these genes facilitate pathogenesis (Soll (1992); Soll(1996)), and they include secreted aspartyl proteinases (Hube et al.(1994); Morrow et al. (1992); Morrow et al. (1993); White et al. (1993))and drug resistance proteins (Balan et al. (1997)). No single phenotypictrait has been found to be solely responsible for pathogenesis.Switching results in antigenic variability on the yeast cell surface;one cell surface antigen has been identified as specific to the opaquephenotype. Reviewed in Soll (1992). Accordingly, switching provides amechanism to enhance pathogenesis by generating phenotypic plasticity.

[0007] With the emergence of drug-resistant Candida strains and agrowing population of immunocompromised individuals, there is anincreasing need to find new methods of treating candidiasis. With thisgoal in mind, there is a need to identify proteins that either areassociated with virulent Candida phenotypes or that are themselvespathogenic factors. Antibodies against these proteins also are expectedto inhibit the pathogenic activity of these proteins, thus contributingto amelioration of the symptoms of candidiasis and preferably to adecrease in mortality associated with systemic infection. For example,vaccines may be directed against proteins associated preferentially withpathogenic phenotypes.

SUMMARY OF THE INVENTION

[0008] The present invention addresses these needs by providing, fromCandida yeast such as C. glabrata, a hemolysin-like protein (HLP) andits encoding polynucleotide. The inventors' discovery of an HLP in C.glabrata led to their identifying a similar C. albicans HLP and encodinggene. The inventors have found that C. glabrata, like C. albicans,undergoes phenotype switching, which regulates the expression of the C.glabrata HLP. HLP expression coincides with hemolytic activity, and itis expected that a phenotype expressing an HLP will have virulentproperties conferred by the hemolytic activity of that HLP. The HLP ofthe invention is expected to exhibit hemolytic activity also because ofstructural similarity to partial (α-type) or complete (β-type)hemolysins identified in a number of other pathogens.

[0009] HLP polypeptides of the invention are antigenic and are useful asvaccine components to raise a protective immune response againstCandida. Further, antibodies directed against a Candida HLP, or anantigenic fragment thereof, are useful in indicating the presence of avirulent phenotype. The antibodies also can be used to protect againstor ameliorate candidiasis associated with a virulent Candida phenotype.In one embodiment, antibodies raised by an HLP protein, or an antigenicfragment thereof, are capable of inhibiting hemolytic activity of anHLP.

[0010] Accordingly, the invention provides a vaccine, comprising:

[0011] (i) an antigenic polypeptide comprising 10 contiguous amino acidresidues of either of the proteins shown in SEQ ID NOS:2 or 4; or

[0012] (ii) a polynucleotide encoding an antigenic polypeptidecomprising 10 contiguous amino acid residues of either of the proteinsshown in SEQ ID NOS:2 or 4, wherein the polynucleotide is operablylinked to a promoter capable of driving transcription in a host cell;and

[0013] (iii) a pharmaceutically acceptable adjuvant or carrier vehicle.

[0014] The polypeptide component of this vaccine alternately maycomprise 12 contiguous amino acids of either of the proteins shown inSEQ ID NOS:2 or 4. Alternately, it may comprise 30 or 40 contiguousamino acids of either of the proteins shown in SEQ ID NOS:2 or 4. In oneembodiment, the carrier vehicle is a protein that is covalentlyconjugated to the polypeptide. In another embodiment, the vaccinecomprises either of the proteins shown in SEQ ID NOS:2 or 4.

[0015] The vaccine alternately may comprise a polynucleotide encoding apolypeptide comprising 10, 12, 30, or 40 contiguous amino acids ofeither of the proteins shown in SEQ ID NOS:2 or 4. In one embodiment,the polynucleotide encodes either of the proteins shown in SEQ ID NOS:2or 4.

[0016] A method of diagnosing a virulent phenotype of Candida comprisesexposing a body fluid from an individual suspected of having a virulentphenotype of Candida with a detectably labeled antibody that is capableof binding an HLP of Candida.

[0017] A method of treatment of an individual infected with a virulentphenotype of Candida comprises administration of a pharmaceuticalcomposition that comprises an antibody that is capable of binding an HLPof Candida. In one embodiment, a method of inducing an immune response,preferably comprising a cellular immune response, comprisesadministering to an individual a pharmaceutical composition thatcomprises either:

[0018] (i) an antigenic polypeptide comprising 10 contiguous amino acidresidues of either of the proteins shown in SEQ ID NOS:2 or 4; or

[0019] (ii) a polynucleotide encoding an antigenic polypeptidecomprising 10 contiguous amino acid residues of either of the proteinsshown in SEQ ID NOS:2 or 4, wherein the polynucleotide is operablylinked to a promoter capable of driving transcription in a host cell;and

[0020] (iii) a pharmaceutically acceptable adjuvant or carrier vehicle.

[0021] Another embodiment of the invention is an isolated polynucleotidethat exhibits greater than about 80% sequence identity to polynucleotidesequence SEQ ID NO:1, or a polynucleotide having the sequence shown inSEQ ID NO:1, or a polynucleotide encoding a polypeptide having thesequence shown in SEQ ID NO:2. Polypeptides encoded by thepolynucleotides are encompassed by the invention, including apolypeptide having the sequence shown in SEQ ID NO:2. Antigenicfragments preferably comprises at least 30 contiguous amino acids of theprotein shown in SEQ ID NO:2.

[0022] BRIEF DESCRIPTION OF THE FIGURES

[0023]FIG. 1: A cloned C. glabrata genomic DNA fragment that is 1532nucleotides in length (SEQ ID NO: 1).

[0024]FIG. 2: The polynucleotide sequence of a C. albicans HLP-encodingDNA, including a promoter region, cloned from C. albicans strain WO-1(SEQ ID NO:3).

[0025]FIG. 3: Comparison of the deduced amino acid sequence of a C.glabrata HLP (“CgHLP”; SEQ ID NO:2) and C. albicans HLP (“CaHLP”; SEQ IDNO:4).

[0026]FIG. 4 : Comparison of five regions with high similarity in bothposition and arrangements of amino acids among a C. glabrata and C.albicans HLP and hemolysins from seventeen bacteria and Caenorizabditiselegans.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] The inventors have identified polynucleotides and the encodedpolypeptides of Candida hemolysin-like proteins (HLPs) and presentlydisclose HLPs from C. albicans and C. glabrata. The inventors havefurther discovered a novel phenotype switching in C. glabrata. C.glabrata reversibly switches among white (Wh), light brown (LB) and darkbrown (DB) colony phenotypes. Switching in C. glabrata, as in C.albicans, is believed to represent a general strategy for thecombinatorial expression of genes encoding proteins involved invirulence. In particular, the C. glabrata HLP is expressedpreferentially in different phenotypes, and, by analogy with C.albicans, the phenotype expressing the HLP at high levels is expected toexpress other virulence factors at high levels.

[0028] Hemolytic activity will play a particularly important role inpathogenesis, especially in systemic infection by Candida, wherecirculating red blood cells are exposed directly to the pathogens. Ithas been postulated that Candida acquires iron that is liberated fromlysed red blood cells of the infected host. Manns et al. (1994).Further, in various ways beyond the outright loss of red blood cells,hemolysis impairs the health of the host and contributes to mortalityfrom systemic infection. Reviewed in Maslow et al. (1999); Traub et al.(1996); May et al. (1996); Hacker et al. (1983). Accordingly, inhibitionof hemolytic activity will be valuable in the treatment of candidiasis.In one embodiment of the invention, antibodies against an HLP inhibithemolytic activity.

[0029] Regardless of whether other virulence factors are produced incombination with HLP, the association of HLP with hemolytic activity is,itself, sufficient to identify a phenotype that expresses high levels ofan HLP as a virulent phenotype. With this in mind, any Candida phenotypethat expresses an HLP is defined as a “virulent phenotype,” for thepurposes of this invention.

[0030] Similarity of C. albicans and C. glabrata HLP with KnownHemolysins

[0031] HLPs of the invention are believed to exhibit hemolytic activity,because phenotypes expressing these HLPs at high levels are hemolytic,and because the HLPs are structurally related to other hemolysinsexhibiting either partial (α-type) or complete (β-type) hemolysisidentified in a number of other pathogens. Fairweather et al. (1983);Helms et al. (1977); Li et al. (1999); Leao et al. (1995). Electronmicroscopy suggests that hemolysins lyse cells by forming oligomericpore complexes through the cellular plasma membrane of the target cell.Nizet et al. (1997). See also Lange et al. (1997).

[0032] A polynucleotide encoding an HLP of the invention was foundserendipitously during in an attempt to clone another gene in C.glabrata. FIG. 1 shows a cloned C. glabrata genomic DNA fragment that is1532 nucleotides in length and encodes an HLP with three putativetrans-membrane domains. The discovery of a hemolysin-like gene in C.glabrata prompted the inventors to investigate the possible presence ofa similar gene in C. albicans. The polynucleotide sequence of an HLPgene in C. albicans is shown in FIG. 2 (SEQ ID NO:2).

[0033] Comparison of the deduced amino acid sequence of a C. albicansand C. glabrata HLP revealed an overall 75% sequence identity spanning351 amino acid residues of the protein. Both the amino-terminal endconsisting of 60-85 amino acid residues and carboxy-terminal endconsisting of over 400 amino acid residues appear unique to eachspecies. See FIG. 3 (SEQ ID NOS:2 and 4).

[0034] Comparison of these two Candida HLPs with known hemolysinsrevealed similarity among a variety of pathogenic and nonpathogenicbacteria as well as eukaryotes. Gish et al. (1993); Worley et al.(1995). These hemolysins range in size from 65-85 kD. Welch (1995);Tweter (1995). In particular, comparison with data bases revealed fiveregions with high similarity in both position and arrangements of aminoacids among a C. glabrata and C. albicans HLP and hemolysins fromseventeen pathogenic microorganisms and Caenorhabditis elegans. FIG. 4.The mean percent identity of region 1 of the deduced C. glabrata proteinsequence and hemolysins from 16 unrelated organisms was 72±12%; percentidentity ranged between 42 and 92%, where “percent identity” refers tothe similarity between sequences based on the BLASTX-BEAUTY sequencealignment algorithm, using the indicated C. glabrata sequence as areference sequence. The mean percent identities of regions 2, 3, 4 and 5were 59±16%, 50±10%, 60±14% and 73±11%, respectively. Furthermore, therelative positions of all five regions in the deduced partial C.glabrata protein were similar to those in the majority of hemolysins ofother organisms. The deduced primary C. glabrata HLP sequence of 508amino acids exhibits 75% identity to the corresponding region of the S.cerevisiae YOL060c gene product.

[0035] A C. albicans hemolytic factor has recently been identified byWanatabe et al. (1999). This factor has been partially characterized andis primarily a polysaccharide, consisting of 95% carbohydrate by weight.It co-migrates with Blue Dextran on a Sephacryl S-100 column, indicatingthat it has an apparent molecular weight of at least 200 kDa, and it isa mannan. It is not believed that the mannan described by Wanatabe etal. is structurally related to the C. albicans HLP shown in SEQ ID NO:4.

[0036] HLP Polypeptides of the Invention

[0037] The phrase “hemolysin-like protein” denotes a polypeptide havingthe amino acid sequence shown in SEQ ID NO:2, which is a C. glabrataHLP, or having the amino acid sequence shown in SEQ ID NO:4, which is aC. albicans HLP. Naturally occurring and synthetically produced variantsof these two proteins also are embraced by “hemolysin-like protein.”“Variants” encompass, for example, naturally occurring HLPs, includingHLPs from other Candida species and allelic variations of theseproteins. Among “variants” in this context also are fragments of theaforementioned proteins that are antigenic when administered with anadjuvant or carrier vehicle. Preferably, fragments are contiguousstretches of amino acids of the proteins of SEQ ID NOS:2 and 4 that are5, 10, 12, 15, 20, 30, or 40 amino acids in length.

[0038] In addition, “variants” further include polypeptides that have amodified amino acid sequence from the aforementioned polypeptides. Theskilled artisan will recognize that structure ultimately definesfunction, and that variants bearing the closest structural relationshipto HLPs shown in SEQ ID NOS:2 and 4 are most likely to preservebiological function and antigenicity. Sequence modifications includeamino acid substitutions, insertions, and deletions. Amino acidinsertions and deletions may be made in the interior of the proteinsequence, as well as at the amino and carboxyl termini. Guidance indetermining which and how many such sequence modifications may be madewithout abolishing biological or antigenic activity may be found usingcomputer programs well known in the art, for example, DNASTAR software(DNASTAR Inc., 1801 Univerity Ave., Madison, Wis. 53705).

[0039] Substitutions preferably are conservative, that is, one aminoacid is replaced with one of similar shape and charge. Conservativesubstitutions are well known in the art and include, for example, thechanges of: alanine to serine; arginine to lysine; asparigine toglutamine or histidine; aspartate to glutamate; cysteine to serine;glutamine to asparigine; glutamate to aspartate; glycine to proline;histidine to asparigine or glutamine; isoleucine to leucine or valine;leucine to valine or isoleucine; lysine to arginine, glutamine, orglutamate; methionine to leucine or isoleucine; phenylalanine totyrosine, leucine or methionine; serine to threonine; threonine toserine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine;and valine to isoleucine or leucine.

[0040] The sequence of variants preferably will have an 80% identity tothe full length proteins shown in SEQ ID NOS:2 and 4. Again, “percentidentity” is determined by the BLASTX-BEAUTY sequence algorithm, wherethe test sequence is aligned and compared to either sequences set forthin SEQ ID NOS:2 and 4. More preferably, variants will have at leastabout 85% identity to these sequences. Even more preferably, the percentidentity will be at least about 90%, and most preferably, the percentidentity will be at least about 95%, or even 98%. Likewise, variants offragments of the proteins of SEQ ID NOS:2 and 4 will be useful for theinvention, for instance, as antigenic fragments. Such variants will haveat least about 85% identity to fragments of the proteins of SEQ ID NOS:2and 4. Even more preferably, the percent identity will be at least about90%, and most preferably, the percent identity will be at least about95%, or even 98%. Preferably, antigenic fragments will be at least 30amino acids in length.

[0041] Additional polypeptide sequences or other moieties, such ascovalently attached, detectable tags, may be added to the proteins ofthe invention. Additional polypeptide sequences may fused to either theamino or carboxyl termini of the polypeptides of the invention, and theymay be useful, for example, in assisting the expression, purification,and/or detection proteins of the invention. For example, these varioussequences include those well known in the art that are useful inpurification of recombinantly expressed proteins. A preferred fusionprotein comprises a “His tag” sequence, which facilitates purificationof the recombinantly expressed protein. A preferred system is the TALONSnondenaturing protein purification kit for purifying 6xHis-taggedproteins under native conditions (CLONTECH, Palo Alto, Calif.).

[0042] “Isolated” polypeptides are not in a naturally occurring formand/or have been purified to remove at least some portion of cellular ornon-cellular molecules with which the proteins are naturally associated.The present HLPs may be isolated by the methodology described byHerbelin et al. (1995) for the purification of hemolysins, for example.

[0043] Assays for Hemolytic Activity

[0044] An HLP is expected to exhibit hemolytic activity, as set forthabove. In one embodiment, an HLP variant exhibits hemolytic activity. Itis expected that a hemolytically active HLP variant will include some orall of the structural elements that are conserved among knownhemloysins, indicated in FIG. 4. The HLPs of the invention bear closestresemblance to hemolysins that exhibit partial (α-type) or complete(β-type) hemolysis. Fairweather et al. (1983); Helms et al. (1977); Liet al. (1999); Leao et al. (1995).

[0045] Hemolytic activity can be tested by any of several routine assayswell known in the art. These assays include inspection of cells exposedto hemolysins by light microscopy, release of detectable cytoplasmicproteins, such as hemoglobin or enzymes, Trypan blue exclusion, or Evansblue-albumin flux. Gibson et al. (1999); Thelestam et al. (1994).Additional assays include a hemolytic plate assay or cell free brothassay as described in Parveen (1992). Alternately, a fluorometric assay,using erythrocyte ghosts, may be used, as described in Serra et al.(1992).

[0046] These assays can be used to test the ability of compounds toinhibit the hemolytic activity of an HLP or an HLP variant. Highthroughput assays, designed around one of the assays described above,for example, can simultaneously test a plurality of candidate compounds.Antibodies will be screened using a hemolytic assay to identify thoseantibodies with inhibitory activity, which will allow the identificationof potentially therapeutically useful antibodies. This screening methodcan also be applied to other candidate molecules, such as drugs, toidentify those with inhibitory activity. Such drugs are expected to beuseful as components of a pharmaceutical composition for treatment ofcandidiasis.

[0047] HLP Polynucleotides of the Invention

[0048] A polynucleotide of the invention encodes any polypeptide of theinvention, and includes all the possible combinations of codons thatencode particular amino acids. Polynucleotides of the invention, inaddition to encoding polypeptides, may be useful as oligonucleotideprobes for the identification of other HLP polynucleotides, as set forthin the Examples below. A polynucleotide comprises at least 5 nucleotidesof a nucleic acid (RNA, DNA, a combination thereof, or analoguesthereof), provided by any means, such as synthetic purification.Polynucleotides having at least about 10 nucleotides are preferred, andpolynucleotides having at least about 20 nucleotides are more preferred.The nucleotide sequence of an HLP gene fragment shown in FIG. 1 (SEQ IDNO: 1) has been deposited in the National Center for BiologicalInformation under accession number AF196836.

[0049] Methods of Isolating Polynucleotides of the Invention

[0050] Identification of polynucleotides encoding HLP in C. glabrata andC. albicans will allow the skilled artisan to identify structurallyrelated ploynucleotides encoding variants of these polynucleotides,other polynucleotides from other Candida species. Methods suitable foridentification of related polynucleotides are well known in the art.

[0051] Three such approaches are described in the Examples, below. Theyare (1) a PCR-based strategy using degenerate primers, (2) the use of anon-hemolytic Saccharomyces cerevisiae system as a screen for genes forCandida hemolytic activity, and (3) a search for HLP homologs in Candidaby “mining” nucleic acid and protein databases, followed by functionaland structural characterization of the suspect genes and gene products.

[0052] Vectors and Host Cells of the Invention

[0053] The invention also provides vectors and host cells that comprisethe polynucleotides of the invention. Host cells include any eukaryotic,prokaryotic, or other cell that is suitable for propagating and/orexpressing an isolated nucleic acid that is introduced into the hostcell by any suitable means known in the art. The cell can be part of atissue or organism, isolated in culture or in any other suitable form.Vectors include any nucleic acid compound used for introducing exogenousnucleic acid into host cells. A vector comprises a nucleotide sequencewhich may encode one or more polypeptide molecules. Plasmids, cosmids,viruses, and bacteriophages, in a natural state or which have undergonerecombinant engineering, are non-limiting examples of commonly usedvectors to provide recombinant vectors comprising at least one desiredisolated nucleic acid molecule. Reviewed in Sambrook et al. (1989) andAusubel et al. (1989). Because of the similarity of the HLP proteins topartial (α-type) or complete (β-type) hemolysis, it is expected thatthey will be functional hemolysins in secreted form. Accordingly,preferred expression vectors comprise any of the well-characterizedsecretion signals.

[0054] A host cell of the invention strain may be chosen for its abilityto post-translationally modify the expressed recombinant protein. Suchmodifications of the polypeptide include, for example, glycosylation andacylation. Post-translational processing which cleaves a nascent form ofthe protein may also be important for correct secretion, folding and/orfunction. Different host cells may be chosen to ensure the correctmodification and processing of the recombinantly expressed protein.

[0055] “Isolated” polynucleotides are removed from their native ornaturally occurring environment. For example, recombinantpolynucleotides within vectors are considered isolated for the purposesof the present invention. Isolated polynucleotides include in vivo or invitro RNA transcripts of the polynucleotides of the invention. Isolatedpolynucleotides further include synthetically produced molecules, wherethe nucleic acid in other than a naturally occurring form. Isolatedpolynucleotides include genomic DNA that has been removed from thechromosome in which it occurs naturally.

[0056] Antibodies and Vaccines of the Invention

[0057] Candida HLPs and antigenic fragments thereof will be useful ascomponents of a vaccine. In one embodiment, a vaccine comprises HLP fromC. albicans or C. glabrata, or antigenic fragments thereof. Particularlyuseful vaccines will raise antibodies that block or inhibit thehemolytic activity of the protein. Such antibodies can be screened invitro, using the various hemolysis assays described above.

[0058] Protection against pathogens by vaccination against a hemolysinantigen has met with considerable success. Furesz et al. (1997); Haga etal. (1997); Byrd et al. (1997); U.S. Pat. No. 6,007,825; U.S. Pat. No.5,731,151. In some instances, the hemolytic activity of a hemolysin wasblocked by polyclonal antibodies. For example, Lange et al. (1997).

[0059] In general, the preparation of polyclonal and monoclonalantibodies, as well as hybridomas capable of producing the desiredantibody, are well known in the art. For example, Campbell (1984); St.Groth et al. (1980); Kohler et al. (1975); Kozbor et al. (1983); Cole etal. (1985).

[0060] i) Polyclonal Antibodies

[0061] Polyclonal antiserum, containing antibodies to heterogenousepitopes of a single protein, can be prepared by immunizing suitableanimals with the expressed protein described above, which can beunmodified or modified, as known in the art, to enhance immunogenicity.Immunization methods include subcutaneous or intraperitoneal injectionof the polypeptide.

[0062] The protein immunogen may be modified or administered in anadjuvant in order to increase the protein's antigenicity. Methods ofincreasing the antigenicity of a protein are well known in the art andinclude, but are not limited to, the inclusion of an adjuvant duringimmunization. Adjuvants include Freund's (complete and incomplete),mineral gels such as aluminum hydroxide, surface active substances suchas lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanin, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and Corynebacteriumparvum.

[0063] HLP antigens alternately may be conjugated to a carrier vehicle,or “immunocarrier,” to improve the interaction between T and B cells forthe induction of an immune response against the antigen. This isparticularly preferable for the production of vaccines inimmunocompromised individuals, who also will be those predisposed tocandidiasis. Preferred carrier vehicles are proteins that are covalentlyconjugated to a HLP polypeptide of the invention. Proteins useful ascarrier vehicles are well known in the art and include tetanus toxoid,diphtheria toxoid, P. aeruginosa exotoxin A, and variants thereof, asdescribed, for example, in Fattom et al. (1993).

[0064] Effective polyclonal antibody production is affected by manyfactors related both to the antigen and to the host species. Forexample, small molecules tend to be less immunogenic than other and mayrequire the use of carriers and/or adjuvant. In addition, host animalresponse may vary with site of inoculation. Both inadequate or excessivedoses of antigen may result in low titer antisera. In general, however,small doses (high ng to low μg levels) of antigen administered atmultiple intradermal sites appears to be most reliable. Host animals mayinclude but are not limited to rabbits, mice, and rats, to name but afew. An effective immunization protocol for rabbits can be found inVaitukaitis et al. (1971). Affinity of the antisera for the antigen maybe determined by preparing competitive binding curves, as described, forexample, by Fisher(1980).

[0065] ii) Monoclonal Antibodies

[0066] Monoclonal antibodies (MAbs) may be obtained by any techniquethat provides for the production of antibody molecules by continuouscell lines in culture or in vivo. MAbs may be produced by makinghybridomas, which are immortalized cells capable of secreting a specificmonoclonal antibody. Monoclonal antibodies to any of the proteins,peptides and epitopes thereof described herein can be prepared frommurine hybridomas according to the classical method of Kohler et al.(1975) and U.S. Pat. No. 4,376,110, or modifications of the methodsthereof. Such modifications include the human B-cell hybridoma technique(Kosbor et al. (1983)) and the EBV-hybridoma technique (Cole et al.(1985)).

[0067] iii) Antibody Derivatives and Fragments

[0068] Fragments or derivatives of antibodies include any portion of theantibody which is capable of binding the target antigen, or a specificportion thereof. Antibody fragments specifically include F(ab′)₂, Fab,Fab′ and Fv fragments. They may be made by conventional recombinant DNAtechniques or, using the classical method, by proteolytic digestion withpapain or pepsin. See Current Protocols in Immunology, chapter 2,Coligan et al., eds., John Wiley & Sons (1991-92). Other antibodyderivatives include single chain antibodies. U.S. Pat. No. 4,946,778;Bird (1988); Huston et al. (1988). Single chain antibodies are formed bylinking the heavy and light chain fragments of the Fv region via anamino acid bridge, resulting in a single chain FV (SCFv).

[0069] Derivatives also include “chimeric antibodies.” Morrison et al.(1984); Neuberger et al. (1984); Takeda et al. (1985). These chimerasare made by splicing the DNA encoding a mouse antibody molecule ofappropriate specificity with, for instance, DNA encoding a humanantibody molecule of appropriate specificity. Thus, a chimeric antibodyis a molecule in which different portions are derived from differentanimal species, such as those having a variable region derived from amurine mAb and a human immunoglobulin constant region. These are alsoknown sometimes as “humanized” antibodies and they offer the addedadvantage of at least partial shielding from the human immune system.They are, therefore, particularly useful in therapeutic in vivoapplications.

[0070] iv) Labeled Antibodies

[0071] The present invention further provides the above-describedantibodies in detectably labeled form. Antibodies can be detectablylabeled through the use of radioisotopes, affinity labels (such asbiotin, avidin, etc.), enzymatic labels (such as horseradish peroxidase,alkaline phosphatase, etc.) fluorescent labels (such as FITC orrhodamine, etc.), paramagnetic atoms, etc. Labeling procedures are wellknown in the art, for example see Sternberger et al. (1970); Bayer etal. (1979); Engval et al. (1972). The labeled antibodies of the presentinvention can be used for in vitro, in vivo, and in situ diagnosticassays.

[0072] v) Immobilized Antibodies

[0073] The foregoing antibodies also may be immobilized on a solidsupport. Examples of such solid supports include plastics such aspolycarbonate, complex carbohydrates such as agarose and sepharose,acrylic resins and such as polyacrylamide and latex beads. Techniquesfor coupling antibodies to such solid supports are well known in theart. Weir et al. (1986). The immobilized antibodies of the presentinvention can be used for in vitro, in vivo, and in situ assays as wellas for immunoaffinity purification of the proteins of the presentinvention.

[0074] vi) DNA Vaccines

[0075] DNA-based immunization refers to the induction of an immuneresponse to an antigen expressed in vivo from a gene introduced into theanimal. This method offers two major advantages over classicalvaccination in which some form of the antigen itself is administered.First, the synthesis of antigen in a self-cell mimics in certainrespects an infection and thus induces a complete immune response butcarries absolutely no risk of infection. Second, foreign gene expressionmay continue for a sufficient length of time to induce strong andsustained immune responses without boost. See U.S. Pat. No. 5,780,448,for example.

[0076] Genes have been introduced directly into animals by using liveviral vectors containing particular sequences from an adenovirus, anadeno-associated virus, or a retrovirus genome. The viral sequencesallow the appropriate processing and packaging of a gene into a virion,which can be introduced to animals through invasive or non-invasiveinfection. Naked DNA transfects relatively efficiently if injected intoskeletal muscle. Wolff et al. (1990). Alternately, plasmid DNA may becoated onto the surface of small gold particles and introduced into theskin by a helium-driven particle accelerator, or “gene-gun.” Pecorino etal. (1992). DNA has also been introduced into animal cells byliposome-mediated gene transfer. DNA-liposome complexes, usuallycontaining a mixture of cationic and neutral lipids, are injectedintravenously or into various tissues or instilled into the respiratorypassages. Nabel et al. (1992).

[0077] Several mammalian animal models of DNA-based immunization againstspecific viral, bacterial or parasitic diseases have been reported. Inmost of these studies, a full-range of immune responses, includingantibody production and a cytotoxic T lymphocyte response, was obtained.See U.S. Pat. No. 5,780,448, for example.

[0078] Diagnostic Methods of the Invention

[0079] A variety of protocols useful for detecting and measuring thepresence of HLPs of the invention in body fluids or tissue and cellextracts, using either polyclonal or monoclonal antibodies specific forthe protein, are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA) and fluorescentactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopesmay be employed. Well known competitive binding techniques may also beemployed. See, e.g., Hampton et al. (1990); Coligan et al. (1997 andperiodic supplements); and Maddox et al. (1983).

[0080] The immunoassays of this invention can be embodied in test kitswhich comprise HLP proteins of the invention or HLP-specific antibodies.Such test kits can be in solid phase or liquid phase formats, and theycan be based on immunohistochemical assays, ELISAs, particle assays,radiometric or fluorometric assays, using, for example, avidin/biotintechnology.

[0081] Therapeutic Methods of the Invention

[0082] A vaccine of the present invention can be administered to asubject who then acts as a source for globulin, produced in response tochallenge from the specific vaccine (“hyperimmune globulin”), thatcontains antibodies directed against an HLP of the invention. U.S. Pat.No. 5,770,208. A subject thus treated would donate plasma from whichhyperimmune globulin would then be obtained, via conventionalplasma-fractionation methodology, and administered to another subject inorder to impart resistance against or to treat Candida infection.Hyperimmune globulins according to the invention are particularly usefulfor immune-compromised individuals, for individuals undergoing invasiveprocedures or where time does not permit the individual to produce hisown antibodies in response to vaccination.

[0083] A method of preparing an immunotherapeutic agent against Candidainfection comprises steps of immunizing subjects with a compositionaccording to the invention, collecting plasma from the immunizedsubjects, and harvesting a hyperimmune globulin that contains antibodiesdirected against Candida from the collected plasma. The hyperimmuneglobulin contains antibodies directed against the HLPs of the invention.An immunotherapy method comprises a step of administering thishyperimmune globulin to a subject.

[0084] Hyperimmune globulins would preferably be administered in thepresence of known compounds that are useful for treating Candida.Amphotericin B is one such compound. Fluconazole has been shown to be aneffective and safe alternative in non-neutropenic patients.5-Fluorocytosine has been used in combination with amphotericin B in thetreatment of deep-seated infections. Liposomal formulations ofamphotericin B and other antifungal drugs may be used. Verduyn Lunel etal. (1999).

[0085] The present invention, thus generally described, will beunderstood more readily by reference to the following examples, whichare provided by way of illustration and are not intended to be limitingof the present invention.

EXAMPLES Example 1 C. glabrata HLP Expression is Associated withPhenotype Switching

[0086] Expression of C. glabrata HLP is regulated by switching, with theorder of transcript levels of the various phenotypes DB>LB>Wh. To verifythat the levels of C. glabrata HLP transcripts were in fact selectivelyregulated in Wh, LB, and DB, the transcript levels of additional controlgenes were analyzed. Transcript levels of various constitutivelyexpressed genes were similar in Wh, LB and DB cells, supporting theconclusion that C. glabrata HLP is selectively regulated by switching.

[0087] The level of HLP transcript in the three switch phenotypes of C.glabrata was assessed by slot blot analysis. The level of transcript waslowest in Wh cells, intermediate in LB cells and highest in DB cells.Densitometric scans provided relative transcript ratios of 1:20:25 forWh:LB:DB.

[0088] The experiments assessing switching and gene expression wereperformed on strain 35B11, an oral isolate from a healthy, elderlyindividual. Lockhart et al. (1999). In order to test whether highfrequency switching was a general characteristic of C. glabrata,switching was tested in three additional C. glabrata isolates, 65FLOP,65TL1 and 75PLI. For each strain, cells from a single colony were firstgrown in YPD medium containing 1 mM CuSO₄, then plated at low density onYPD agar containing 1 mM CuSO₄ and colony phenotypes assessed after fivedays of incubation at 25° C. In every case, multiple phenotypes based oncolony color (Wh, LB, DB) were observed at frequencies roughly similarto those observed for strain 35B11. Thus, switching is a generalcharacteristic of C. giabrata strains. Experiments summarizing thefrequency of switching among white, light brown and dark brown colonyphenotypes in C. glabrata and switching in three additional pathogenicisolates of C. glabrata are summarized in Tables I and II. TABLE 1Frequency of alternative phenotypes in white, light brown and dark browncolonies in C. glabrata. Frequency Frequency Dark Light FrequencyFrequency Original Number Brown Brown White Sectored Phenotype CloneColonies Colonies Colonies Colonies Colonies* Light 1 1234 3 × 10⁻² — 3× 10⁻³ 4 × 10⁻¹ Brown 2 1425 2 × 10⁻² — 6 × 10⁻³ 4 × 10⁻¹ (LB) 3 1833 3× 10⁻² — 7 × 10⁻³ 3 × 10⁻¹ 4 1223 1 × 10⁻² — <10⁻³ 5 × 10⁻¹ mean 2 ×10⁻² 4 × 10⁻³ 1 × 10⁻¹ (±s.d.) (±1 × 10⁻²⁾ (±3 × 10⁻³⁾ (±1 × 10⁻¹⁾ Dark1 6418 — 8 × 10⁻³ <2 × 10⁻⁴  3 × 10⁻¹ Brown 2 2897 — 3 × 10⁻⁴ 1 × 10⁻³ 1× 10⁻³ (DB) 3 2719 — 9 × 10⁻³ 4 × 10⁻⁴ 2 × 10⁻³ mean 3 × 10⁻³ 5 × 10⁻⁴ 2× 10⁻³ (±s.d.) (±5 × 10⁻³⁾ (±5 × 10⁻⁴⁾ (±1 × 10⁻³⁾ White 1 792 4 × 10⁻¹6 × 10⁻² — 6 × 10⁻¹ (Wh) 2 725 4 × 10⁻¹ 7 × 10⁻² — 6 × 10⁻¹ 3 1509 4 ×10⁻² 5 × 10⁻² — 2 × 10⁻¹ 4 938 2 × 10⁻² 3 × 10⁻² — 5 × 10⁻² 5 1422 5 ×10⁻² 4 × 10⁻² — 9 × 10⁻² mean 2 × 10⁻¹ 5 × 10⁻² 3 × 10⁻¹ (±s.d.) (±2 ×10⁻¹⁾ (±2 × 10⁻²⁾ (±3 × 10⁻¹⁾

[0089] TABLE 2 Switching in three additional pathogenic isolates of C.glabrata. Frequency Original Number Variant Strain Phenotype Colonies WhLB DB Sectored Colonies 65FLOP LB 3775  5.8 × 10⁻³* — 5.8 × 10⁻³    9%†1.2 × 10⁻² 65TL1 LB 2330 4.0 × 10⁻⁴ — 5.4 × 10⁻² >80%† 5.5 × 10⁻² 75PL1DB 3290 2.4 × 10⁻³ 6.0 × 10⁻⁴ —  0.4%‡ 3.0 × 10⁻³

Example 2 PCR-based Strategy Using Degenerate Primers to Identify HLPHomologues

[0090] Degenerate primers may be designed that are complementary tostretches of DNA encoding highly conserved domains of HLP proteins.Exemplary highly conserved domains include regions 1 and 3 of the C.albicans HLP indicated in FIG. 2. Region 1: CaHLP-N: 5′-ATR GTS RTS TTRGGT GAA ATR ATR CC-3′ Region 3: CaHLP-C1: 5′-GGD GTC ATS ATN TCN TNRAC-3′ CaHLP-C2: 5′-CCA TGG ACA DTC CCA GTR GAM-3′

[0091] Using specific combinations of forward and reverse primers, PCRreactions may performed using either total genomic DNA or total RNA poolfrom Candida hypha cultures. The amplifications may be performed usingeither classical Taq polymerase (Life Technologies, Inc, Gaithersburg,Md.) or high fidelity Long PCR kit (Roche Biochemicals Inc.,Indianapolis, Ind.). The cycle conditions used will be 92° C. for 1 min,36° C. for 1 min and 68° C. for 90 sec and 40 cycles. As compared to theclassical Taq polymerase, high fidelity long PCR protocol will be morereproducible and will result in consistent amplified products ranging insize from 250 bp to 500 bp. The pool of PCR products will be cloned intopGEM-T easy vector for sequence analysis. The PCR insert library will bescreened and sequenced to reveal clones that encode hemolysin-likegenes. Once it is confirmed that the insert contains sequences for aputative hemolysin gene, the full-length gene will be cloned usingtechniques well known in the art. The regulation of the gene byphenotype switching and the function of the gene product can then bedetermined.

Example 3 Using Non-hemolytic Saccharomyces cerevisiae System to Screenfor Genes for Candida hemolytic Activity

[0092]S. cerevisiae has been used extensively to identify Candida geneseither based on functional complementation of homologous genes orfunctional induction of new activities associated with heterologousnon-homologous genes. Boone et al. (1991); Gillum et al. (1984);Rosenbluh et al. (1985); Fu et al. (1998); Gaur et al. (1997).

[0093] The main principle of functional complementation is to identifyCandida HLP polynucleotides by gain of function analysis of cellstransformed by either genomic or cDNA libraries. Two research groupshave recently demonstrated the power of this experimental strategy. Twogenes, ALS1 and ALA1, whose products are involved in adherence of C.albicans to host cells, were identified by introducing the C. albicansgenomic library into a S. cerevisiae strain and assaying transformantsfor adherence to endothelial cells (Fu et al. (1998)) or to magneticbeads coated with extracellular matrix proteins (ECMs) (Gaur et al.(1997)). We describe below the strategy for the isolation of genes thatencode hemolytic activity of C. albicans.

[0094] A genomic library of Candida will be constructed in a 2μ-based S.cerevisiae shuttle vector (PEMBLYe30) with Leu2 as a selectable markerand will be used in the study. A 2μ-based genomic library was chosenrather than a centromere based genomic library because the former systemwill exhibit greater sensitivity for identifying hemolytic factor due tohigher plasmid copy number per cell resulting in production of higherlevels of hemolytic activity.

[0095] As the first step in this strategy, a Leu2⁻ S. cerevisiae strainwill be transformed with a Candida genomic library using lithium acetatetransformation protocol. Schiestl et al. (1989). Followingtransformation, cells will be spread on selective SD agar plates withoutleucine. After 4 days of growth at 30° C., the total number oftransformants will be scored. Approximately 10,000 to 25,000transformants representing 6 to 8 genomic equivalents (considering theaverage size of DNA inserts to be 5 to 7 Kb) will be included in thehemolytic activity screen. The individual transformants will be pickedand collected in 20 to 25 pools, each pool consisting of approximately1000 clones. From each pool, approximately 1000 individual cells will bespread on blood agar plates containing human blood (Manns et al., 1994).The plates will be incubated at 30° C. for 3 to 4 days and periodicallymonitored for hemolytic zones surrounding the colonies. As a control forbackground, transformants containing the parental plasmid pEMBLYe30 willalso be monitored for hemolytic activity. Those transformants exhibitinga zone of hemolytic activity will be selected for further analysis. Theplasmid DNA containing Candida putative genes for hemolytic activitywill be isolated from the select S. cerevisiae transformants andtransformed into E. coli. Both the integrity of the parental plasmid andthe size of insert DNA will be determined by agarose gelelectrophoresis. The next step will be to confirm that the isolatedplasmid DNA does contain the gene for hemolytic activity observed inprimary transformants. This will be accomplished by transforming theoriginal S. cerevisiae strain, with each of the plasmid DNAs containingputative hemolysin-like gene, and analysis of transformants forhemolysis on human blood agar (Manns et al., 1994). After reconfirmingthat the putative clones harbors the Candida genes for hemolyticactivity, plasmids will be transferred into E. coli for amplification.The high quality plasmid DNAs will be purified from E. coli and verifiedfor the presence of insert DNA similar to the original insert in theprimary transformants. We will then determine the complete nucleotidesequence of the selected clones. The derived nucleotide and amino acidsequences will be compared to DNA and protein data bases using Blast andBeauty software. Gish (1993); Worley et al. (1995). After confirmingthat the DNA fragment encodes the hemolytic activity, we will analyzethe regulation of the gene and the physiological role of the geneproduct as described below:

[0096] 1) NORTHERN BLOT. Northern blot analysis of total RNA will beperformed to determine whether the hemolysin gene is regulated at thetranscriptional level during the white-opaque transition, the bud-hyphatransition and/or by environmental parameters such as temperature, pHand constituents of growth medium. The translational product ofhemolysin gene will be analyzed by using specific epitope-taggedhemolysins.

[0097] 2) LOSS OF FUNCTION. Gene knockout analysis will be done usingthe strain TS3.3, an ura3⁻ derivative of WO-1. The homozygous deletionmutants of the hemolysin gene(s) will be analyzed to determine thecontribution of hemolysin genes in virulence in both skin and systemicanimal models of infection. Kvaal et al. (1997).

[0098] 3) MISEXPRESSION. If the expression of hemolytic activity isdetermined to be phase-specific (Kvaal et al., 1997), the misexpressionof hemolysin genes will be analyzed.

[0099] 4) DETERMINATION OF FUNCTIONAL DOMAINS. Protein domains requiredfor hemolytic activity in strains harboring homozygous deletion for thegene will be identified by hemolytic activity on human blood agar.Initially, we will determine the domains or regions that are essentialfor hemolytic activity. Next, we will map critical amino acid residuesby targeted point mutations of specific amino acid residues based onstructure-functional studies of bacterial hemolysins.

[0100] 5) PROTEIN MODIFICATION. First, we will establish whetherhemolysin is modified by glycosylation. If the hemolysin isglycosylated, we will then determine whether glycosylation is requiredfor hemolytic activity by using specific glycosylation inhibitors suchas tunicamycin and specific anion transport inhibitors such as DIDS,SITS and BS (Watanabe et al., 1999). The role of identifiedcarbohydrate-modified amino acids in hemolysis will be addressed bycreating point mutations at specific amino acid residues.

Example 4 Search for HLP Homologs by “Mining” Nucleic Acid and ProteinDatabases

[0101] Known sequences from Candida HLPs may be compared to other knownnucleotide sequences on various databases. For example, a searchrevealed three nucleic acids in Stanford's C. albicans genome database,con4-2646, con4-2740 and con4-2796, which were highly homologues to theHLP of C. glabrata. Identification of sequences on the basis of homologywill then be followed by structural and functional characterizationusing the methods described above.

Example 5 Miscellaneous Methods of the Invention

[0102] Yeast isolates and maintenance. C. glabrata isolates may beobtained from any source, such as from blood of infected individuals.Isolates were typed as C. glabrata by sugar assimilation pattern and byhybridization to the C. glabrata-specific probes Cg6 and Cg12 (21).Clones were stored at room temperature on a YPD agar slant (2% glucose,2% Bacto Peptone, 1% yeast extract, 2% agar, Difco Laboratories,Detroit, Mich.) in a capped tube. The switch phenotypes were propagatedon YPD agar plates containing 1 mM CuSO₄ at 25° C. Each of thephenotypes may also be stored at −80° in glycerol.

[0103] Measurements of phenotypic switching. To assess the frequency ofvariant phenotypes in a clonal population of C. glabrata, cells from asingle 3 day old colony exhibiting a homogenous colony phenotype wereinoculated into YPD liquid medium containing 1 mM CuSO₄ and were grownat 25° C. for approximately 6 to 8 hr to a density of 5×10⁶ cells perml. Cells then were diluted and were plated at a density ofapproximately 50 cells per agar plate. Plates were incubated at 25° C.for 5 days and the colony phenotypes were scored.

[0104] Growth kinetics. Cells from a 3 day old single colony exhibitinga homogeneous phenotype were inoculated into 10 ml of YPD liquid mediumcontaining 1 mM CuSO₄ in a 30 ml test tube and incubated until theconcentration reached 1×10⁷ cells per ml. Then 5×10⁶ cells wereinoculated into a 250 ml Erlenmyer flask, containing 50 ml of fresh YPDliquid medium plus 1 mM CUSO₄, and were rotated at 25° C. for 48 hr.Samples were removed every 2 hr over a 48 hr period and were vortexed.The concentration of spheres was measured in a hemocytometer.

[0105] PCR amplification of C. glabrata and C. albicans HLP gene. PCRproducts were generated in 100 μl of a reaction mixture containing 10 mMBuffer B (Fischer Scientific, St. Louis, Mo.), 1.2 mM MgCl₂, 100 μMdNTP, 50 muM dNTP, 50 μM of the 5′ primer and 50 μM of the 3′ primer,and 2.5 units of Taq polymerase (Fischer Scientific). Conditions for PCRcycling included 40 cycles of denaturation at 92° C. for 1 min,annealing at 40° C. for 1.5 min, and extension at 68° C. for 1.5 min.

[0106] PCR products were gel-purified and used as template forgenerating radioactive probes. The C. glabrata HLP PCR product wasobtained using primers SLF1-N5′ ATGTCATCGCAAAACCTCAAT3′ and SLF1-C5′CTGCCTGCTAATTTCACCTTG3′. The PCR product was cloned in E. coli andsequenced in both directions using an ABI model 373A automaticsequencing system and fluorescent Big dye terminator chemistry (PerkinElmer/Applied Biosystems Inc., Fostor city, CA). The alignment ofnucleotide sequences and comparison with sequences in the databases wereperformed with the BLASTX-BEAUTY analysis program.

[0107] DNA fingerprinting and southern blot analysis. DNA fingerprintingwas performed as described in Schmid et al. (1990) with complex DNAfingerprinting probes (Lockhart et al. (1997)). In brief, total genomicDNA from each of the C. glabrata switch phenotypes was prepared by themethod of Scherer et al. (1987). Approximately 1 μg of total genomic DNAwere digested with EcoRI (4 U/μg of DNA) and the resulting fragmentselectrophoresed at 35 V for 15 hr in a 0.65% (w/v) agarose gel. DNA wastransferred by capillary blotting to Hybond N⁺nylon membrane (AmershamParmacie Biotech, Buckinghamshire, England), hybridized with randomlyprimed [³²P] dCTP-labeled probe, and autoradiographed as described inSchmid et al. (1990). For Southern blot analyses performed for purposesother than DNA fingerprinting, DNA was digested with SalI, the digestionfragments were resolved in a 0.8% (w/v) agarose gel, and the Southernblots were hybridized with randomly primed [³²P] dCTP-labeled probes.

[0108] Slot blot and northern analysis of transcripts. Total cellularRNA was isolated by methods described in Srikantha et al. (1995) withthe following modifications: pellets of 3×10⁸ washed cells from 3 dayold colonies were frozen, mixed with an equal volume of acid-washedglass beads (400 μm diameter) and 450 μl of RNA extraction buffer from aRNAeasy Mini Kit (Qiagen Inc., Valencia, Calif.), and agitated with abead beater device (Biospec Products, Bartlesville, Okla.). Two μg oftotal cell RNA were immobilized on a Zetabind Nylon membrane (CUNO,Inc., Meriden, Conn.) using the slot blot apparatus PR48 (HoeferPharmacia Biotech. Inc., San Francisco, Calif.), hybridized withrandomly primed ³²P-labeled probe, and autoradioraphed. Hybridizationintensities were compared by scanning the slot blots with the“Densitometry of Lanes” option of the DENDRON software package version2.0 (SolltechInc., Iowa City, Iowa). To perform successivehybridizations of the slot blot with different probes, the previousprobe was stripped using standard methods. Northern blot hybridizationwas performed according to methods described in Kvaal et al. (1997).

[0109] The description, specific examples, and data, while indicatingexemplary embodiments, are given by way of illustration and are notintended to limit the present invention. Various changes andmodifications within the present invention will become apparent to theskilled artisan from the disclosure, and thus are considered part of theinvention.

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What is claimed is:
 1. A vaccine, comprising: (i) an antigenicpolypeptide comprising 10 contiguous amino acid residues of either ofthe proteins shown in SEQ ID NOS:2 or 4; or (ii) a polynucleotideencoding an antigenic polypeptide comprising 10 contiguous amino acidresidues of either of the proteins shown in SEQ ID NOS:2 or 4, whereinsaid polynucleotide is operably linked to a promoter capable of drivingtranscription in a host cell; and (iii) a pharmaceutically acceptableadjuvant or carrier vehicle.
 2. The vaccine of claim 1, wherein thevaccine comprises said polypeptide, and wherein said polypeptidecomprises 12 contiguous amino acids of either of the proteins shown inSEQ ID NOS:2 or
 4. 3. The vaccine of claim 1, wherein the vaccinecomprises said polypeptide, and wherein said polypeptide comprises 30contiguous amino acids of either of the proteins shown in SEQ ID NOS:2or
 4. 4. The vaccine of claim 1, wherein the vaccine comprises saidpolypeptide, and wherein said polypeptide comprises 40 contiguous aminoacid residues of either of the proteins shown in SEQ ID NOS:2 or
 4. 5.The vaccine of claim 1, wherein the vaccine comprises said polypeptide,and wherein said carrier vehicle is a protein that is covalentlyconjugated to said polypeptide.
 6. The vaccine of claim 1, wherein thevaccine comprises said polynucleotide, and wherein said polynucleotideencodes a polypeptide comprising 10 contiguous amino acids of either ofthe proteins shown in SEQ ID NOS:2 or
 4. 7. The vaccine of claim 1,wherein the vaccine comprises said polynucleotide, and wherein saidpolynucleotide encodes a polypeptide comprising 12 contiguous aminoacids of either of the proteins shown in SEQ ID NOS:2 or
 4. 8. Thevaccine of claim 1, wherein the vaccine comprises said polynucleotide,and wherein said polynucleotide encodes a polypeptide comprising 30contiguous amino acid residues of either of the proteins shown in SEQ IDNOS:2 or
 4. 9. The vaccine of claim 1, wherein the vaccine comprises theprotein shown in SEQ ID NO:2.
 10. The vaccine of claim 1, wherein thevaccine comprises the protein shown in SEQ ID NO:4.
 11. A method ofdiagnosing a virulent phenotype of Candida, comprising exposing a bodyfluid from an individual suspected of having a virulent phenotype ofCandida with a detectably labeled antibody that is capable of binding anHLP of Candida.
 12. A method of treatment of an individual infected witha virulent phenotype of Candida, comprising administration of apharmaceutical composition that comprises an antibody that is capable ofbinding an HLP of Candida.
 13. A method of inducing an immune response,comprising administering to an individual a pharmaceutical compositionthat comprises: (i) an antigenic polypeptide comprising 10 contiguousamino acid residues of either of the proteins shown in SEQ ID NOS:2 or4; or (ii) a polynucleotide encoding an antigenic polypeptide comprising10 contiguous amino acid residues of either of the proteins shown in SEQID NOS:2 or 4, wherein said polynucleotide is operably linked to apromoter capable of driving transcription in a host cell; and (iii) apharmaceutically acceptable adjuvant or carrier vehicle.
 14. The methodof claim 13, wherein the immune response is a cellular immune response.15. An isolated polynucleotide that exhibits greater than about 80%sequence identity to polynucleotide sequence SEQ ID NO:
 1. 16. Thepolynucleotide of claim 15, wherein said polynucleotide has the sequenceshown in SEQ ID NO:1.
 17. The polynucleotide of claim 15, wherein saidpolynucleotide codes for a polypeptide having the sequence shown in SEQID NO:2.
 18. An isolated polypeptide encoded by the polynucleotideaccording to claim
 15. 19. The polypeptide of claim 18, wherein saidpolypeptide has the sequence shown in SEQ ID NO:2.
 20. An antigenicfragment of the polypeptide according to claim 19, wherein said fragmentcomprises at least 30 contiguous amino acids of the protein shown in SEQID NO:2.